**3. Transfer mechanisms of antibiotic resistance genes**

For antibiotics to function and inhibit microbial growth, they must be at the proper concentration so that they can cross the cellular wall and interact with their target. As previously mentioned, AR is the capacity that has a microorganism to resist the inhibitory activity of an antibiotic beyond the normal susceptibility of similar bacterial species [27]. On the other hand, the different mechanisms of AR are based on the modification of the antibiotic target site as well as on the reduction of the antibiotic concentration that manages to get the cell target.

LAB are considered carriers of resistance genes that could propagate their genes within the food chain between food and humans, as well as to the environment through different mechanisms [27–30]. According to the FAO and WHO [24], it is important to determine whether starter or probiotic cultures intended for human or animal consumption have mobile resistance genes that could be transferred to other microorganisms [6, 31]. In addition, some authors have demonstrated that the use of antibiotics in animals destined for consumption, either as growth promoters or pathogen inhibitors, is directly related to the presence of AR microbiota in the human gastrointestinal tract [27, 32]. On the other hand, Gad et al. [21] isolated some *Lactobacillus*, *Streptococcus*, and *Lactococcus* strains from both pharmaceutical and probiotic dairy products, but the AR tests from the pharmaceutical probiotic isolates were free of resistance genes, unlike the LAB isolated from dairy products that showed resistance profiles comparable to those from pathogens such as *Staphylococcus spp*., *Escherichia coli*, and *Salmonella spp*. Furthermore, some *Enterococcus faecium* strains have demonstrated the transference of vancomycin resistant genes from to *Lactobacillus acidophilus* La5 "in vitro" and "in vivo" studies in the gut mice [33].

Exposure to antibiotics may allow bacteria to develop different mechanisms to counteract the bactericidal effect; a single bacterium can develop different types of resistance; these systems include an intrinsic or innate and the acquired resistance mode. Among these, the mechanism that prevails within bacteria varies according to the nature of the antibiotic, the target site, the bacterial species, and/or whether the resistance gene is part of the chromosome or mobile elements such as plasmids or transposons [12, 19, 28].

#### **3.1. Mechanisms of resistance in LAB**

effect, reduction in the serum cholesterol level, antioxidant effect, protection against colon cancer, reduction in the allergy symptoms, reduction in dental caries, and reduction in the obesity index [21, 22]. In addition, secondary metabolites with health-promoting properties include the antihypertensive angiotensin-converting enzyme produced through the proteolytic system of

LAB are considered naturally resistant to several antibiotics and may have the potential to acquire resistance to other antimicrobials or to disseminate the resistance to pathogens present in the gastrointestinal tract of animals and humans [9]. For instance, Shao et al. [11] demonstrated that two isolates of *L. plantarum* possessed the *aaad*A and *ant*(6) genes associated to the resistance to streptomycin, and the overexposure to this antibiotic dramatically increased the minimum inhibitory concentration (MIC) and increased a cross-resistance to other antibiotics from the same class. On the other hand, the presence of 6% strains isolated from some pharmaceutical and dairy products from Egypt with tetracycline [*tet*(M)] and/ or erythromycin [*erm*(B)] resistant genes has been reported [21]. In a similar study, a high incidence of *Lactobacillus* resistant to vancomycin (58%), erythromycin (10.8%), tetracycline (4.3%), gentamicin (48%), and ciprofloxacin (26%) was reported in Turkish fermented dairy products [13]. However, studies made by Flores and Mayo [25] indicate that no transfer of the tetracycline [*tet*(M)] and erythromycin [*erm*(B)] resistant genes from *S. thermophilus* to *L. delbrueckii* was detected during the production and storage of yogurt. Furthermore, the food chain can facilitate the transmission of antibiotic-resistant bacteria between animals, foods, and humans, being the fermented milk and meat products the most common vehicle for antibiotic-resistant bacteria to the indigenous flora of the gastrointestinal tract, as these products are consumed without a thermal treatment [12]. Even though that some reports confirm the transmission of resistant determinants, the two most common resistant genes in LAB are tetracycline [*tet*(M)] and erithromycin [*erm*(B)] resistant genes, followed by *cat* genes coding for chloramphenicol resistance [26]. Considering the wide range of potential applications of LAB in the industry and in the human and animal health, there is a need of their detailed

*Lactobacillus helveticus*, *Lactobacillus acidophilus*, and *Lactobacillus delbrueckii* [14, 22].

58 Antimicrobial Resistance - A Global Threat

examination that involves the detection of AR genes.

**3. Transfer mechanisms of antibiotic resistance genes**

For antibiotics to function and inhibit microbial growth, they must be at the proper concentration so that they can cross the cellular wall and interact with their target. As previously mentioned, AR is the capacity that has a microorganism to resist the inhibitory activity of an antibiotic beyond the normal susceptibility of similar bacterial species [27]. On the other hand, the different mechanisms of AR are based on the modification of the antibiotic target site as well as on the reduction of the antibiotic concentration that manages to get the cell target.

LAB are considered carriers of resistance genes that could propagate their genes within the food chain between food and humans, as well as to the environment through different mechanisms [27–30]. According to the FAO and WHO [24], it is important to determine whether starter or probiotic cultures intended for human or animal consumption have mobile resistance genes that could be transferred to other microorganisms [6, 31]. In addition, some authors have demonstrated that the use of antibiotics in animals destined for consumption, either as growth Two relevant elements must be present for the antibiotic-target interaction, first the antibiotic must recognize the target, and the concentration of the antibiotic in the target must be sufficient to inhibit the bacterial growth. A resistance mechanism conduces to the antibiotic failure to inhibit the bacterial growth due to an inefficient antibiotic-target interaction, which can be classified as passive and active. The passive mechanism can only be transferred to other cells by clonal transfer that involves modifications of the target site or decrease in antimicrobial absorption, without affecting the antibiotic structure; this resistance is also known as intrinsic resistance. In contrast, the active mechanism involves the reduction on the concentration of the intracellular antibiotic by modification or degradation of its structure with enzymes or through the action of efflux pumps [34, 35].

**Figure 1** shows the mechanisms by which some bacteria can show resistance to antibiotics that involves (1) modification of the antibiotic by enzymatic complexes that prevent the antibiotic-target interaction, (2) enzymatic degradation of intra- or extracellular antibiotics, and (3) reduction in the intracellular antibiotic concentration through the activation of flow pumps or due to the change in the cell wall permeability [19].

The main mechanism of resistance to antibiotics presented by LAB has been related with multidrug-resistant (MDR) efflux pumps involved in the expulsion of structurally unrelated compounds [31, 36]. Wacher-Rodarte et al. [37] analyzed LAB isolated from pozol (a traditional fermented maize beverage), identifying that MDR strains such as *Lactococcus lactis* and *Lactobacillus plantarum* present active efflux pumps, including the chromosomally encoded ABC type with the LmrA transporter (*lmrA* gene). On the other hand, Poelarends et al. [38] demonstrated that the presence of the LmrA transporter in *Lactococcus lactis* is associated with the innate resistance of 17 up to 21 clinically relevant antibiotics, including aminoglycosides (kanamycin and gentamicin), lincosamines (clindamycin), macrolides (erythromycin), quinolones (ciprofloxacin), and tetracyclines. Other authors such as Casado Muñoz et al. [39] reported that *Lactobacillus pentosus* and *Leuconostoc pseudomesenteroides* isolated from fermented olives are resistant to cephalosporins, streptomycin, and kanamycin due to the

in traditional and industrialized fermented foods although they have intrinsic resistance to quinolones (ciprofloxacin and nalidixic acid), mupirocin, tetracyclines, and aminoglycosides such as streptomycin; however, all the genes are located in the chromosome with a limited transference to other genus [28, 43]. It has been reported that some LAB genera have intrinsic resistance to bacitracin, vancomycin, kanamycin, teicoplanin, and quinolones [28]. This

Antibiotic Resistance in Lactic Acid Bacteria http://dx.doi.org/10.5772/intechopen.80624 61

• Modification of the cell wall, commonly observed in the resistance to glycopeptides (vancomycin and teicoplanin) and non-ribosomal antibiotics (bacitracin). In particular, *Lactobacillus plantarum* and *Enterococcus faecium* present innate resistance to vancomycin, due to the substitution of D-alanine residues of the muramyl pentapeptide cell wall by D-lactate (high-level resistance) or D-serine (low-level resistance) in the chemical structure

• Enzymatic inactivation such as for aminoglycosides (neomycin, kanamycin, streptomycin) or quinolones (ciprofloxacin, norfloxacin, nalidixic acid) prevents the binding of these antibiotics with their specific targets, as observed for *Lactobacillus* and *Enterococcus* for the 16S rRNA of the 30S ribosomal bacterial subunit and DNA gyrase, respectively, that explains

Extrinsic or acquired resistance is one in which bacteria can incorporate into their cellular structure mobile genetic material capable of conferring resistance to certain antibiotics. Unlike intrinsic resistance, the acquired resistance is only found in some traits or bacterial subpopulations. The gene propagation may occur between bacteria of different genera or between different organisms. The horizontal gene transfer (HGT) occurs when the bacteria is capable of acquiring new genes that can increase their intrinsic resistance spectrum, or they can transfer resistance to other microorganisms or directly to humans or animals, which is already considered a health risk, according to the WHO. Therefore, the protocols for the analysis of resistance genes in LAB are increasing as they have a high capacity to acquire AR and since they have a close relationship with food processing [6, 19, 31, 45, 46]. **Figure 2** shows the three main mechanisms of HGT; some of which are not considered relevant in the transfer of resistance to antibiotics in LAB, for example, transduction (through bacteriophages) and transformation (when DNA is released from one bacterium and is absorbed by another), as the conjugation is the primary mechanism observed in lactic acid bacteria [12, 19, 47, 48].

The conjugation is the transfer of mobile genetic material from plasmids or transposons through a tube of proteins, called sexual pilus [6]. Plasmids are extrachromosomal DNA molecules capable of autonomous replication and that may confer resistance to microorganisms against antibiotics and represent one of the main mobile elements for dissemination of antibiotic-resistant genes against β-lactams, aminoglycosides, tetracyclines, chloramphenicol,

Plasmids have a large number of genetic determinants that may confer resistance by conjugation, and it is important to consider that a single bacterium can have multiple plasmids [49].

sulfonamides, trimethoprim, macrolides, and quinolones [29, 47, 48].

of the peptidoglycan, thus avoiding the antibiotic interaction [35, 41, 44].

the intrinsic resistance to both groups of antibiotics [29, 40].

*3.1.2. Extrinsic resistance*

intrinsic resistance mechanisms presented by LAB include:

**Figure 1.** Mechanisms of antibiotic resistance in the LAB: (1) enzymatic modification, (2) enzymatic degradation, and (3) enzyme efflux pumps. Adapted from Sharma et al. [19].

variation of the cell wall permeability as their main mechanism of resistance; they also pointed that both strains presented a complex AcrAB-TolC system involved in MDR efflux pumps for β-lactams, fluoroquinolones, chloramphenicol, tetracycline, and other genes related with chromosomally encoded superfamily pumps *norA* and *Mde* that confer resistance to chloramphenicol and fluoroquinolones.

The resistance to aminoglycosides in LAB has not been reported, although in recent years LAB isolated from farm origin show resistant to gentamicin, kanamycin, and streptomycin, whose resistance mechanism is associated to impaired transport or enzymatic inactivation by three main aminoglycoside-modifying enzymes (AMEs) as N-acetyltransferases (AACs), O-phosphotransferases (APHs), and O-nucleotidyltransferases (ANTs) encoded by MGEs (mobile genetic elements) like transposons and insertion sequences [40].

Some bacteria belonging to the genera *Enterococcus*, *Lactobacillus*, *Pediococcus*, and *Bifidobacterium* present both intrinsic or innate and extrinsic or acquired AR, which can be a factor of food safety as they can spread resistance to other bacteria by vertical (between species) or horizontal transference (between bacterial genera) [25, 29, 31, 41].

#### *3.1.1. Intrinsic resistance*

Intrinsic resistance is the natural or innate ability of a bacterium to survive the effect of antibiotics, as a result of mutations derived from changes in the bacterial physiological state or by the uncontrolled exposure to antibiotics [42]. Intrinsic resistance has a minimum propagation potential between bacterial genera, as resistance genes are located into the chromosome with a limited transference to other genus, which represents a low risk within nonpathogenic bacteria. Any gene responsible for intrinsic resistance could be disseminated and transferred to other bacteria if it is flanked by insertion sequences that may promote its mobilization [12]. For instance, *Bifidobacterium* strains are commonly used as starter cultures and/or prebiotics in traditional and industrialized fermented foods although they have intrinsic resistance to quinolones (ciprofloxacin and nalidixic acid), mupirocin, tetracyclines, and aminoglycosides such as streptomycin; however, all the genes are located in the chromosome with a limited transference to other genus [28, 43]. It has been reported that some LAB genera have intrinsic resistance to bacitracin, vancomycin, kanamycin, teicoplanin, and quinolones [28]. This intrinsic resistance mechanisms presented by LAB include:


#### *3.1.2. Extrinsic resistance*

variation of the cell wall permeability as their main mechanism of resistance; they also pointed that both strains presented a complex AcrAB-TolC system involved in MDR efflux pumps for β-lactams, fluoroquinolones, chloramphenicol, tetracycline, and other genes related with chromosomally encoded superfamily pumps *norA* and *Mde* that confer resistance to chloram-

**Figure 1.** Mechanisms of antibiotic resistance in the LAB: (1) enzymatic modification, (2) enzymatic degradation, and (3)

The resistance to aminoglycosides in LAB has not been reported, although in recent years LAB isolated from farm origin show resistant to gentamicin, kanamycin, and streptomycin, whose resistance mechanism is associated to impaired transport or enzymatic inactivation by three main aminoglycoside-modifying enzymes (AMEs) as N-acetyltransferases (AACs), O-phosphotransferases (APHs), and O-nucleotidyltransferases (ANTs) encoded by MGEs

Some bacteria belonging to the genera *Enterococcus*, *Lactobacillus*, *Pediococcus*, and *Bifidobacterium* present both intrinsic or innate and extrinsic or acquired AR, which can be a factor of food safety as they can spread resistance to other bacteria by vertical (between species) or horizon-

Intrinsic resistance is the natural or innate ability of a bacterium to survive the effect of antibiotics, as a result of mutations derived from changes in the bacterial physiological state or by the uncontrolled exposure to antibiotics [42]. Intrinsic resistance has a minimum propagation potential between bacterial genera, as resistance genes are located into the chromosome with a limited transference to other genus, which represents a low risk within nonpathogenic bacteria. Any gene responsible for intrinsic resistance could be disseminated and transferred to other bacteria if it is flanked by insertion sequences that may promote its mobilization [12]. For instance, *Bifidobacterium* strains are commonly used as starter cultures and/or prebiotics

(mobile genetic elements) like transposons and insertion sequences [40].

tal transference (between bacterial genera) [25, 29, 31, 41].

phenicol and fluoroquinolones.

60 Antimicrobial Resistance - A Global Threat

enzyme efflux pumps. Adapted from Sharma et al. [19].

*3.1.1. Intrinsic resistance*

Extrinsic or acquired resistance is one in which bacteria can incorporate into their cellular structure mobile genetic material capable of conferring resistance to certain antibiotics. Unlike intrinsic resistance, the acquired resistance is only found in some traits or bacterial subpopulations. The gene propagation may occur between bacteria of different genera or between different organisms. The horizontal gene transfer (HGT) occurs when the bacteria is capable of acquiring new genes that can increase their intrinsic resistance spectrum, or they can transfer resistance to other microorganisms or directly to humans or animals, which is already considered a health risk, according to the WHO. Therefore, the protocols for the analysis of resistance genes in LAB are increasing as they have a high capacity to acquire AR and since they have a close relationship with food processing [6, 19, 31, 45, 46]. **Figure 2** shows the three main mechanisms of HGT; some of which are not considered relevant in the transfer of resistance to antibiotics in LAB, for example, transduction (through bacteriophages) and transformation (when DNA is released from one bacterium and is absorbed by another), as the conjugation is the primary mechanism observed in lactic acid bacteria [12, 19, 47, 48].

The conjugation is the transfer of mobile genetic material from plasmids or transposons through a tube of proteins, called sexual pilus [6]. Plasmids are extrachromosomal DNA molecules capable of autonomous replication and that may confer resistance to microorganisms against antibiotics and represent one of the main mobile elements for dissemination of antibiotic-resistant genes against β-lactams, aminoglycosides, tetracyclines, chloramphenicol, sulfonamides, trimethoprim, macrolides, and quinolones [29, 47, 48].

Plasmids have a large number of genetic determinants that may confer resistance by conjugation, and it is important to consider that a single bacterium can have multiple plasmids [49].

In particular, the enterococcal and *Lactobacillus* genera may be associated to a health risk, as they carry innate and acquired resistance genes and because of their high residence in food and

Antibiotic Resistance in Lactic Acid Bacteria http://dx.doi.org/10.5772/intechopen.80624 63

Enterococci are widely distributed in vegetables, dairy products, prepared foods, and meat products and used as probiotics; however, they have intrinsic resistance to a large number of antibiotics such as β-lactams and aminoglycosides. In some cases, they can present profiles of resistance similar to enterococci considered nosocomial emergent pathogens which could present multiple drug resistance (MDR) with mechanisms of resistance that include modification of pharmacological targets, inactivation of therapeutic agents, overexpression of efflux pumps, and sophisticated

adaptive response of cell envelope that promotes survival in the human host [41, 52].

in the gastrointestinal microbiome of humans and animals [28, 36].

**Table 2.** Lactic acid bacteria resistant to antibiotics isolated from food [20, 37, 49, 50].

*3.2.1. Enterococcus*

**Figure 2.** Mechanisms of horizontal gene transfer in the LAB. (1) conjugation is a process requiring the cell to cell contact via cell surface pili; (2) cell transformation by integrating extracellular DNA; (3) transduction, bacteriophages may transfer bacterial DNA from a previously infected donor cell to the recipient cell. Adapted from Sharma et al. [19] and Von Wintersdorff et al. [47].

Some authors indicate that the genetic diversity of resistance is proportional to the number of plasmids present in the environment, without forgetting that there are other mobile elements such as transposons and integrons, although these elements do not self-replicate and must be transported by an appropriate plasmid or phage [49, 50]. Some conjugative transposons used as vehicle of antibiotic resistance genes in LAB include Tn*916*, Tn*918*, Tn*920*, Tn*925*, Tn*2702 (E. faecalis*), Tn*5233* (*E. faecium*), Tn*5276*, and Tn*5301 (Lactococcus lactis*) [19].

#### **3.2. Resistance to antibiotics in LAB**

As mentioned, the presence of resistance genes in LAB is considered a public health problem, so the EFSA through the panel of additives and products or substances used in animal feed (FEEDAP) developed a technical guide to identify the bacteria that show acquired resistance to antibiotics such as ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracyclines, and chloramphenicol [51]. Most LAB that present acquired resistance in the food production chain include the obligate homofermentative *Lactobacillus* genera (*L. helveticus, L. acidophilus, L. delbrueckii*), obligate heterofermentative *Lactobacillus* (*L. reuteri*, *L. fermentum*), heterofermentative *Lactobacillus* facultative (*L. plantarum*, *L. rhamnosus*, *L. paracasei*), *Lactococcus lactis*, *Streptococcus thermophilus*, *Pediococcus spp*., *Leuconostoc spp*., and *Enterococcus spp*. [31, 51]. On the other hand, LAB can be incorporated into food in the form of probiotic or starter cultures or they can be part of the natural microbiota of traditional fermented foods, but some authors have found that the vast majority of these bacteria are resistant to antibiotics [6, 19, 31, 40, 45]. **Table 2** shows some AR LAB isolated from traditional fermented foods, industrialized and probiotic recommended for improving the intestinal microbiota [20, 37, 49, 50]. In particular, the enterococcal and *Lactobacillus* genera may be associated to a health risk, as they carry innate and acquired resistance genes and because of their high residence in food and in the gastrointestinal microbiome of humans and animals [28, 36].

#### *3.2.1. Enterococcus*

Some authors indicate that the genetic diversity of resistance is proportional to the number of plasmids present in the environment, without forgetting that there are other mobile elements such as transposons and integrons, although these elements do not self-replicate and must be transported by an appropriate plasmid or phage [49, 50]. Some conjugative transposons used as vehicle of antibiotic resistance genes in LAB include Tn*916*, Tn*918*, Tn*920*, Tn*925*, Tn*2702* 

**Figure 2.** Mechanisms of horizontal gene transfer in the LAB. (1) conjugation is a process requiring the cell to cell contact via cell surface pili; (2) cell transformation by integrating extracellular DNA; (3) transduction, bacteriophages may transfer bacterial DNA from a previously infected donor cell to the recipient cell. Adapted from Sharma et al. [19] and

As mentioned, the presence of resistance genes in LAB is considered a public health problem, so the EFSA through the panel of additives and products or substances used in animal feed (FEEDAP) developed a technical guide to identify the bacteria that show acquired resistance to antibiotics such as ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracyclines, and chloramphenicol [51]. Most LAB that present acquired resistance in the food production chain include the obligate homofermentative *Lactobacillus* genera (*L. helveticus, L. acidophilus, L. delbrueckii*), obligate heterofermentative *Lactobacillus* (*L. reuteri*, *L. fermentum*), heterofermentative *Lactobacillus* facultative (*L. plantarum*, *L. rhamnosus*, *L. paracasei*), *Lactococcus lactis*, *Streptococcus thermophilus*, *Pediococcus spp*., *Leuconostoc spp*., and *Enterococcus spp*. [31, 51]. On the other hand, LAB can be incorporated into food in the form of probiotic or starter cultures or they can be part of the natural microbiota of traditional fermented foods, but some authors have found that the vast majority of these bacteria are resistant to antibiotics [6, 19, 31, 40, 45]. **Table 2** shows some AR LAB isolated from traditional fermented foods, industrialized and probiotic recommended for improving the intestinal microbiota [20, 37, 49, 50].

*(E. faecalis*), Tn*5233* (*E. faecium*), Tn*5276*, and Tn*5301 (Lactococcus lactis*) [19].

**3.2. Resistance to antibiotics in LAB**

Von Wintersdorff et al. [47].

62 Antimicrobial Resistance - A Global Threat

Enterococci are widely distributed in vegetables, dairy products, prepared foods, and meat products and used as probiotics; however, they have intrinsic resistance to a large number of antibiotics such as β-lactams and aminoglycosides. In some cases, they can present profiles of resistance similar to enterococci considered nosocomial emergent pathogens which could present multiple drug resistance (MDR) with mechanisms of resistance that include modification of pharmacological targets, inactivation of therapeutic agents, overexpression of efflux pumps, and sophisticated adaptive response of cell envelope that promotes survival in the human host [41, 52].


**Table 2.** Lactic acid bacteria resistant to antibiotics isolated from food [20, 37, 49, 50].

Streptomycin was the first aminoglycoside reported for which resistance appeared in enterococcal strains (concentrations higher than 2000 μg/mL); this resistance is carried out by adenylation of streptomycin, by the action of the enzyme streptomycin adenyltransferase, encoded by the *aadA* gene [35, 41]. Resistance to gentamicin, kanamycin, neomycin, and netilmicin (aminoglycosides as well) is mainly due to the production of the bifunctional enzyme 2′-phosphotransferase-6′ acetyltransferase, which promotes the ATP-dependent phosphorylation of aminoglycosides [41].

bacteriophages [27, 47–49]. The transfer of resistance genes by HGT initiates from the farm animals that were treated with antibiotics used as growth promoters to prevent diseases, but these uncontrolled treatments may induce resistance in their intestinal microbiota; later this biota can reach foods and finally being transferred to the human [3]. Conjugation in food matrices has been reported from commensal bacterium (*Enterococcus faecalis* and *Lactococcus lactis*) to potentially pathogenic strains (*Listeria spp*., *Salmonella spp*., *Staphylococcus aureus*, and *E. coli*) in fermented milk [25, 27]. Also, the transfer of tetracycline resistance genes among LAB has been reported in fermented milk and fermented sausages [27]. Martínez and Baquero [34] report the HGT of tetracycline and vancomycin resistance genes in *Enterococcus faecalis* during the fermentation process of cheese and sausages. Bonham et al. [30] have demonstrated that aged cheeses contain AR *Lactobacillus* and *Lactococcus* that acquired the resistance through HGT induced by the strong condition of microbial selection during the food production and maturation process. A wide diversity of AR species can be found in the human gastrointestinal tract that could be acquired AR genes by HGT; this fact is related to the metagenomic comparison showing that most resistance genes found in the human microbiome are those associated with approved antibiotics used in livestock, which supported the hypothesis that resistance genes can be transferred from the farm to consumers [48]. Therefore, the WHO indicates that the HGT genes can be a significant health problem, as most antibiotic resistance is acquired through the HGT [1].

Antibiotic Resistance in Lactic Acid Bacteria http://dx.doi.org/10.5772/intechopen.80624 65

The FDA categorizes microorganisms with the GRAS distinction after being evaluated in general aspects of safety, taxonomy, potential to produce pathogenicity toxins, resistance to antibiotics, and the historical background of food safety. LAB have a broad history of use in fermented foods and usually recognized as safe. However, the dissemination of AR genes puts the GRAS category in another context, especially for bacteria that present mobile genes of transfer such as *Lactobacillus*, since in the US there are still no guidelines that contemplate the type of resistance in microorganism used in food processing [57]. On the other hand, the EU commission regulates the safety of LAB used as starter or probiotic cultures in the European continent, through the EFSA that establishes guidelines for assigning qualified presumption of safety quality to the organisms since 2003. As previously mentioned, the term QPS is based on reasonable and qualified evidence to allow certain restrictions and may be analogous to the GRAS concept but with more rigid guidelines in which the reliable safety of the bacteria is verified, making clear the phrase "from farm to fork" [58]. The QPS status is given to a bacterium, by the EFSA BIOHAZ Panel (Biological Hazards) that must take into account the following aspects (**Figure 3**): (1) the identity of the taxonomic unit at the genus level; (2) documentation related to the LAB safety, based on scientific evidence and history of use; (3) pathogenicity, in which it is evaluated if any species of the genus has pathogenicity factors, if the information is available, the pathogenic strains are excluded; and (4) knowledge of the final use of the microorganism, identifying if the bacteria is part of the food chain or if

The list of QPS includes species of *Lactobacillus sakei*, *Lactobacillus curvatus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus brevis*, *Lactobacillus rhamnosus*, *Lactobacillus* 

**4. Regulation of the use of LAB**

it is used to produce other products [6, 58].

Strains of enterococci of clinical origin between 60 and 65% exhibit resistance to tetracyclines, although these antibiotics are not routinely used in the treatment of infections caused by these microorganisms. There are two fundamental mechanisms of resistance to tetracyclines in enterococci: flow pumps and protection of the ribosome, thus preventing the binding of the antibiotic. The *tetK* and *tetL* genes code for proteins associated to flow pumps responsible to remove the antibiotic outside of the cell, while the *tetM*, *tetO*, and *tetS* genes code for proteins that provide resistance to tetracyclines for ribosome protection. The *tetL* and *tetM* genes are the most frequent in the chromosome and mobile determinants [41, 52, 53]. Finally, vancomycin (glycopeptide) is the main cause of concern, since this antibiotic is considered at the last option for antibiotic therapy for the treatment of Gram-positive bacteria. The resistance to vancomycin in enterococci is varied, having described six genotypes called *vanA*, *vanB*, *vanC*, *vanD*, *vanE,* and *vanG,* where the genotype *vanA* is more frequent in the *Enterococcus* genus [41].

### *3.2.2. Lactobacillus*

In general, Lactobacilli have a high natural resistance to vancomycin, bacitracin, cefoxitin, metronidazole, nitrofurantoin, and sulfadiazine, as well as antibiotics that inhibit the synthesis of proteins such as chloramphenicol, erythromycin, quinupristin/dalfopristin, lincomycin, clindamycin, and tetracyclines [45]. Guo et al. [54] observed 85% of incidence of vancomycin resistance in food isolated *Lactobacillus* strains, especially in *Lactobacillus plantarum* and *Lactobacillus casei*, with the lower frequency for *Lactobacillus helveticus*, but these resistances are not transferable, as genes are located in the chromosome [54]. In addition, genes that code for resistance to tetracycline and erythromycin have been detected in different *Lactobacillus* species isolated of probiotics and foods [12, 31, 55].

The genus *Lactobacillus* is an excellent receptor for exogenous genes by conjugation, as demonstrated by Abriouel et al. [45] for the conjugative pAMβ1 plasmid found in *Lactobacillus plantarum* that could be obtained from enterococci and streptococci. *Lactobacillus* are commonly susceptible to antibiotics, such as penicillins (ampicillin, oxacillin, and piperacillin), inhibitors of β-lactamase, and cephalosporins (cephalothin and cefuroxime, ceftriaxone and cefoxitin), but in recent years some authors have reported resistance to penicillin G in some strains of *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Lactobacillus plantarum* [45, 56]. Other studies demonstrated that *Lactobacillus rhamnosus* is safe to use as a starter or probiotic culture, despite having resistance genes to vancomycin, as these resistance is encoded into the chromosome [45, 48, 54].

#### **3.3. Horizontal transfer of LAB to the intestinal microbiota**

The horizontal gene transfer (HGT) involves the gene interchange between different bacteria through mobile DNA elements such as plasmids, conjugative transposons, integrons, and bacteriophages [27, 47–49]. The transfer of resistance genes by HGT initiates from the farm animals that were treated with antibiotics used as growth promoters to prevent diseases, but these uncontrolled treatments may induce resistance in their intestinal microbiota; later this biota can reach foods and finally being transferred to the human [3]. Conjugation in food matrices has been reported from commensal bacterium (*Enterococcus faecalis* and *Lactococcus lactis*) to potentially pathogenic strains (*Listeria spp*., *Salmonella spp*., *Staphylococcus aureus*, and *E. coli*) in fermented milk [25, 27]. Also, the transfer of tetracycline resistance genes among LAB has been reported in fermented milk and fermented sausages [27]. Martínez and Baquero [34] report the HGT of tetracycline and vancomycin resistance genes in *Enterococcus faecalis* during the fermentation process of cheese and sausages. Bonham et al. [30] have demonstrated that aged cheeses contain AR *Lactobacillus* and *Lactococcus* that acquired the resistance through HGT induced by the strong condition of microbial selection during the food production and maturation process.

A wide diversity of AR species can be found in the human gastrointestinal tract that could be acquired AR genes by HGT; this fact is related to the metagenomic comparison showing that most resistance genes found in the human microbiome are those associated with approved antibiotics used in livestock, which supported the hypothesis that resistance genes can be transferred from the farm to consumers [48]. Therefore, the WHO indicates that the HGT genes can be a significant health problem, as most antibiotic resistance is acquired through the HGT [1].
